Starch is a natural storage carbohydrate found in a large variety of plants. Starch containing crops are important agricultural crops in almost every part of the world. Among the most important crops containing starch can be mentioned corn, wheat, rice, barley and potatoes.
Enzymatic degradation of starch is part of many industrial processes including brewing, production of glucose or high fructose syrups and production of drinking or fuel ethanol.
In its natural state starch is quite resistant against degradation by many enzymes, and therefore industrial enzymatic degradation of starch is traditionally initiated by a heating step where starch is gelatinized, which renders the starch more sensitive to many enzymes. Gelatinization leads to a high increase in viscosity which may give technical difficulties but is usually necessary in order to obtain a satisfactory high degree of degradation.
A combination of endo-acting enzymes, e.g. α-amylase; exo-acting enzymes, e.g. β-amylase and glucoamylase; and debranching enzymes; e.g. pullulanases and iso-amylases are used for enzymatic degradation of starch.
WO 98/16633 describes hybrids comprising a starch degrading enzyme, a carbohydrate binding domain (CBD) and a linker. Pullulanase is mentioned as an example of a starch degrading enzyme. The only exemplified hybrid consists of an alpha-amylase a CBD and a linker.
EP 1200552 B1 discloses the expression in plants of starch altering proteins, e.g. pullulanases, as hybrids with a starch binding domain (SBD). The hybrids have the benefit that they are localized to the starch granules in the plants and thereby altered starch is produced.
Van Bueren et al. Biochemistry 2004, 43: 15633-15642 describe a four module protein having pullulanase activity isolated from the hyperthermophile eubacteria Thermotoga maritima. One of the four modules of the protein was identifies as a new type carbohydrate binding domain having highest affinity for alpha-(1-4) linked glucans.
N. Sauvonnet et al. 1995. J. Bact. 177 (18): 5238-5246, disclose a study relating to a bacterial pullulanase where it was found that minor insertions could be accepted, fusions of larger proteins to the N- or C-terminal could usually not be secreted efficiently.
WO 2005/096804 discloses polynucleotides for expression in plants. The polynucleotide may encode a fusion polypeptide comprising a first polypeptide and a second peptide. The first polypeptide may have pullulanase activity, and the second peptide may be an N-terminal signal sequence from a starch binding domain.